The vast majority of newly acquired HIV infections develop following exposure to HIV infectivity at mucosal surfaces. Oral exposure to semen from HIV seropositive donors can lead to primary HIV infection. Tonsil organ culture systems have been developed in order to investigate the cellular and molecular mechanisms that underlie HIV transmission in the oral cavity. By using semen from HIV-infected donors as a source of HIV infectivity, it is possible to construct an authentic model of HIV transmission across mucosal surfaces. The major goal of this proposal is to apply HIV infectivity in semen (HIV virions or HIV-infected cells) to intact tonsillar epithelial surfaces to track the dispersal of HIV infectivity immediately after exposure and then monitor the establishment of primary HIV infection. Novel procedures have been established for time-lapse confocal microscopy with live tissue that allow direct visualization of events occurring at, and immediately below the exposed epithelial surfaces. A comprehensive representation of HIV transmission can be derived by combining confocal microscopy with high resolution single-cell in situ hybridization and immunocytochemical detection methods, performed with transverse sections cut from the same tissues. Clear understanding of the cellular and molecular events that support HIV transmission at exposed mucosal surfaces, and recognition of changes induced by semen, will contribute significantly towards the development of protective strategies to reduce the overall frequency of HIV transmission.